The family of NADPH oxidase enzymes serve to generate reactive oxygen species (ROS). The prototype, the phagocyte-type NADPH oxidase produces large amounts of ROS that serve a host defense role. The absence or mutation of components of this oxidase (NOX2/gp91phox, p47phox, p67phox, p22phox, Rac2) results in chronic granulomatous disease (CGD). Also, polymorphisms in p22phox are associated with increased risk of cardiovascular disease. Expression of NADPH oxidase activity, however, is not restricted to phagocytes. Fibroblasts, B lymphocytes and platelets express NOX2/gp91phox protein which leads to ROS production. Recent discovery of multiple homologues of gp91phox (e.g., NOX1, NOX4, Duox1/2) has broadened the NADPH oxidase activity in "non-phagocytic" cells. In non-phagocytic cells and tissues, NOX family members produce lower levels of ROS and are predicted to participate in regulatory and signaling roles and may promote transformation. We have recently shown that T cells express a phagocyte-type NADPH oxidase, and the absence of NADPH oxidase protein components leads to a deficiency in TCR stimulated ROS generation and altered T cell responses. Functionally, T cells from NADPH oxidase- deficient mice or humans exhibited enhanced ERK activation and a relative increase in T helper type 1 cytokine secretion. Therefore, in AIM 1 we will analyze how the phagocyte NADPH oxidase is activated in T cells and assess if the TCR signals serve as "priming" signals for the oxidase. In AIM 2, we will advance our preliminary data, which suggest that T cells also express the calcium dependent, non-phagocytic oxidase Duoxl. Duoxl produces ROS early in TCR signaling and we will analyze how it is activated and its role in regulating TCR signal transduction. Finally, in AIM 3, we will investigate the how the phagocyte NADPH oxidase affects T cell development and function, including activation, apoptosis and TH1/TH2 cytokine production. We will use adoptive transfer of oxidase-deficient T cells into Rag(-/-) recipient mice to examine how the absence of NADPH oxidase activity in only the T cells affects immune responses. The results of this study will characterize the activation and potential role(s) of NADPH oxidase(s) in T cells. It will also suggest how oxidative stress affects T cell signal transduction and development and indicate how T cell function may be altered in patients lacking NADPH oxidase function.